The long ternn goal of this grant proposal is to provide evidence that a low molecular weight G-protein, RhoA, serves a nodal role in protecting cardiomyocytes from stress. Stimuli that activate cardiac RhoA include sphingosine-1-phosphate (SIP) and other ligands for a subset of G-protein coupled receptors (GPCRs) that couple to the G12/13 family of G-proteins. These ligands are generated in response to ischemic stress and inflammation and can stimulate multiple pleiotropic effects of RhoA which protect the heart from damage. Using mouse models with gain and loss of function for RhoA we identified a mechanism for acute protection against ischemia reperfusion injury involving phosphorylation of protein kinase D and a cytoskeletal protein, cofilin2, that translocates to mitochondria. RhoA signaling also leads to transcriptional gene regulation, providing an additional mechanism for protection against more chronic cardiac stress. We have also observed direct effects of RhoA on mitochondrial morphology and autophagy. Three specific aims elucidate these pathways and their physiological roles. Aim #1 examines the effects of cardiac RhoA expression or deletion in a more chronic ischemic model, myocardial infarction (Ml); identifies the SIP3 receptor subtype as the mediator of RhoA signaling and protection using SIP3 receptor KO mice and subtype selective drugs; determines whether ischemic injury is accentuated in mice deleted for G12/13 ;and identifies other cardiomyocyte GPCRs that activate RhoA. Aim #2 examines activation of transcription factors regulated downstream of RhoA (MRTF-A, YAP, NFkB) in WT vs RhoA. TG and KO mice exposed to chronic stress (TAC, Ml); the role of the matricellular integrin binding protein CCN1, an MRTF-A and YAP regulated gene, in hypertrophy decompensation and Ml injury using cardiac specific CCN1 KO mice; other genes will also be identified as potential mediators of salutary or deleterious effects of RhoA activation. Aim #3 will examine the relationship between RhoA mediated regulation of cofilin2 and its effects on mitochondrial function, Bax translocation and cell death. Other studies will examine effects of RhoA localized to mitochondria on the stability of PINK, ubiquitation of mitochondrial proteins and development of mitophagy, another pathway for cardiomyocyte protection. The biomedical significance of this work is in elucidating pathways elicited by myriad GPCR agonists or interventions that activate RhoA in the heart. Understanding which receptors activate these pathways and their downstream molecular mechanisms would provide the basis for therapeutic interventions to selectively enhance salutary vs. deleterious effects of RhoA.